Abstract Although an efficient prophylactic vaccine is available for hepatitis B virus (HBV), chronic hepatitis B (CHB) affects up to 350 million people worldwide, and despite advances remains mostly incurable. In adults, acute HBV infection is cleared in 95% of cases by CD8+ T cell-mediated mechanisms, but chronic, lifelong infection ensues in the remaining individuals, which can lead to liver cirrhosis and cancer. The induction and maintenance of HBV-specific T cells in the liver of infected individuals by traditional vaccine methods has been a challenge due to immunological tolerance, one of the hallmarks of CHB. To overcome these challenges, we will use a CMV vector platform to provide persistent antigen presentation capable of recruiting an expanded set of new HBV-specific T cell populations that are expected to circumvent this problem. CMV-vectored HBV vaccines have several unique features. 1) They can be programmed to elicit conventional (MHC-I restricted) and unconventional (MHC-II and MHC-E-restricted) CD8+ T cells that recognize a larger number of epitopes than traditional vaccines; 2) CMV vectors will elicit and maintain high frequencies of non-exhausted effector memory T cells in the blood and liver; 3) CMV vectors overcome pre-existing anti-CMV-immunity by evading vector-specific immune responses; 4) Use of defined attenuations maintain the immunological induction profile; 5) Our ?epigraph?-algorithm-based antigen design accounts for global genotype variations by integrating data from over 3000 worldwide HBV sequences. We hypothesize that CMV-based HBV immunotherapy will lead to control and immunologic cure of CHB due to the recruitment of novel HBV-specific CD8+ T cells. Our goal is to test this hypothesis in chronically infected humans, in which we expect that vaccination with either one or two injections will result in lifelong immune control of HBV. In the Phase 1 of this Fast Track Program we will first design, construct and characterize a spread-deficient HCMV vector expressing HBV antigens with global epitope coverage. We will insert two complementary HBV ?episensus? antigens into a safety-enhanced HCMV- vector through BAC recombineering to generate the final HCMV/HBV construct that can be taken forward for clinical development. In the Phase 2 program we will generate a pre-master virus seed stock ready for GMP production and optimize the manufacturing process of HCMV/HBV vectors. We will characterize the HCMV/HBV vaccine with respect to stability upon multiple passages, maintenance of antigen expression and genomic integrity. We will then characterize the vaccine and the HBV-specific T cell responses in non-human primates (NHP). We will immunize NHP with HCMV/HBV to determine the effective dose, the timing, and the magnitude of the antigen-specific T cell response. We will further determine the breadth and strain-specificity of peripheral and liver T cells upon necropsy using peptides representative of different HBV genotypes. Upon completion of these Aims we will have generated and characterized a novel immunotherapy for HBV that is ready for GMP manufacture and for human safety and efficacy studies.